JP2009131273A - Protease for activating blood clotting factor vii - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new FVII activating protease, and use of the same. <P>SOLUTION: A protease and its proenzyme for activating the blood clotting factor VII is described which (a) is inhibited by the presence of aprotinin, (b) is increased in its activity by calcium ions and/or heparin or heparin-related substances, and (c) in SDS-PAGE, on subsequent staining in the non-reduced state, has one or more bands in the molecular weight range from 50 to 75 kDa and in the reduced state has one or more bands in the molecular weight range from 10 to 35 kDa. The protease and the proenzyme activating the blood clotting factor VII is obtained by previously subjecting blood plasma or prothrombin complex (PPSB) concentrates to an anion exchange chromatography and then subjecting to an affinity chromatography using heparin or heparin analogous substance or dextran sulfuric acid. The invention includes qualitative and quantitive detection of the protease or its proenzyme, and test system for diagnostic application and pharmaceutical preparation comprising an appropriate amount of protease and/or its proenzyme activating the blood clotting factor VII for solving clotting containing fibrin. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a protease for the activation of blood coagulation factor VII, a method for isolating, detecting and inactivating it and a pharmaceutical formulation comprising this protease.

  The blood clotting system is composed of two different cascade-like pathways that activate blood clotting factors present in plasma. Depending on the induction mechanism, the intrinsic or extrinsic pathway is preferentially used to initiate blood clotting.

  When tissue is injured, thromboplastin (tissue factor, TF and phospholipid) is exposed by cells affected as a starter of the extrinsic clotting pathway. Membrane-localized thromboplastin can bind both blood coagulation factor VII (FVII) and circulating, activated FVII (FVIIa). In the presence of calcium ions and lipids, this TF-FVIIa complex leads to FX binding, which is converted to its activated form (FXa) by limited proteolysis. FXa then activates prothrombin to form thrombin, leading to the formation of fibrin, which ultimately results in wound closure.

  Further activation of thromboplastin-bound FVII occurs initially by a self-tactile reaction, but especially after the start of the clotting cascade, it is supported by FXa and thrombin, leading to a marked enhancement of the reaction cascade.

  Administration of FVIIa or FVIIa-containing concentrate has been applied to certain clinical conditions. For example, the so-called FVIII alternative pathway activity (FEIBA) of FVIIa is used in patients affected by hemophilia A and developing antibodies to FVIII as a result of FVIII administration. According to the findings at present, FVIIa is well tolerated in this connection, which does not produce a thrombotic tendency to ensure that a limited but adequate degree of clotting occurs. Is suitable. Recombinant FVIIa has already been used therapeutically and prophylactically. FVII isolated from plasma can also be used after being activated. Proteases such as thrombin can be used for this activation, however, these proteases can themselves strongly activate clotting and can pose a risk of thrombosis. For this reason, subsequent thrombin removal or inactivation is necessary, resulting in loss. As a consequence of the associated thrombus risk, in many cases the use of FXa or FIIa (thrombin) is contraindicated and is applied only in the event of an emergency, for example in the context of significant blood loss and non-hemorrhagic bleeding.

  FVIIa is found only in very low concentrations in the plasma of healthy people. Little is known to date about the formation and origin of FVIIa circulating in the blood. Traces of thromboplastin expressed or released upon cell destruction may play a role in this connection. Factor XIIa is known to cause FVII activation under certain conditions, for example, but the physiological relevance of this reaction has not yet been clarified.

  Surprisingly, a FVII-activating protease different from all previously known proteases has now been discovered in connection with the fractionation of human plasma and certain prothrombin complex concentrates. Examination of this protease showed it to exhibit particularly high amidolytic activity against the peptide substrate S2288 (HD-Isoleucil-L-prolyl-L-arginine-pNA) from ChromogenixAB, Sweden. A special property of this protease is that its amidolytic activity is effectively inhibited by aprotinin. Other inhibitors such as antithrombin III / heparin complexes are also suitable for its inhibition. On the other hand, its activity is increased by heparin and heparin related substances such as heparin sulfate or dextran sulfate and calcium ions. Finally, it was found that this protease can convert FVII to FVIIa in a manner dependent on time and its concentration. This reaction is also inhibited by aprotinin.

Therefore, part of the subject matter of the present invention is
a) inhibited by the presence of aprotinin,
b) its activity is increased by calcium ions and / or heparin or heparin-related substances, and c) in SDS-PAGE, one or more in the molecular weight range of 50-75 kDa in subsequent non-reducing staining In the reduced state, it shows one band with a molecular weight of 40-55 kDa and one or more bands with a molecular weight of 10-35 kDa.
It is a protease that activates blood coagulation factor VII.

The protease concentration and FVIII inactivation when protease and FVIII are incubated for a certain period of time are shown. Inactivation of FVIII is shown when the protease concentration is constant and FVIII is incubated for various times. Fig. 2 shows inactivation of FVIII when incubated with varying concentrations of calcium in the mixture of protease and FVIII.

  In the following specification, the activated form of the protease is referred to as “protease” and the inactivated form is referred to as “proenzyme”.

  This protease was further investigated and experienced a rapid loss of activity after concentration or isolation, which was observed in a solution at pH 7.5 containing 20 mM tris, 0.15 M NaCl. The addition of 0.1% albumin also did not prevent the protease activity from dropping to 50% after 1 hour at room temperature. On the other hand, very good stabilization of this protease was observed in a solution buffered to pH 6.5 with 50 mM sodium citrate. When the solution was adjusted to pH 4-7.2, preferably pH 5.0-7.0, no or very little loss of activity was observed without the addition of any particular stabilizer to the protease solution. However, it is advisable to add stabilizers to the solution, and suitable stabilizers include, in addition to citrate, in particular glutamate, amino acids such as arginine, glycine or lysine, calcium ions and sugars such as glucose, arabinose or The amount of mannose is 1 to 200 mmol / L, preferably 5 to 100 mmol / L. Effective stabilization is also achieved by the addition of 5-80% by weight of a glycol such as ethylene glycol or glycerol, preferably 10-60% by weight is used. In that case, the pH value of the stabilized solution must be 4-9.

  The novel protease, and also its proenzyme, can be obtained by recombinant DNA methods or, for example, by production in the milk of suitable transgenic animals, in particular by fractionation of plasma or prothrombin complex (PPSB) concentrates Can do. The starting material is then first subjected to anion exchange chromatography followed by affinity chromatography of the eluate. For affinity chromatography, heparin or a heparin related substance such as heparin sulfate or dextran sulfate immobilized on a matrix is particularly suitable. When using such chromatographic methods, new proteases and / or proenzymes are selectively bound and can then be eluted again using known methods. The use of spacers can be recommended to couple ligands to Matrux. A heparin-lysine matrix has been found to be particularly suitable for the isolation of novel proteases.

  In SDS-PAGE and subsequent staining, the protease isolated by this method shows one to several bands present in close proximity in the molecular weight range of 55-75 kDa in the non-reducing state. After the reduction, one to several bands were observed in the molecular weight range of 15 to 35 kDa, and one band was observed at 40 to 55 kDa. An additional band of 60-65 kDa, after scanning and quantitative evaluation, made up 5-10% of the total protein and was shown to have non-activated proenzymes. This result was supported by appropriate studies using monoclonal antibodies against this protease. Therefore, it was concluded that the proenzyme of this protease can also be prepared, sterilized and used by the method of the present invention. Part of the subject of the present invention is therefore a proenzyme of the protease that activates blood coagulation factor VII. The ratio of proenzyme is indicated by the 60-65 kDa band. Examples of suitable physiological activators of proenzymes correspond to the amino acid sequences constituting the activation region of proenzyme, thrombin, kallikrein or FVIIa, depending on their substrate specificity.

  Some of the properties of the novel protease described above, namely the fact that it can be isolated from plasma or plasma-derived prothrombin complex (PPSB) concentrate, inhibition of its amidolytic activity by aprotinin and reduced and non-reduced states The above-described electrophoretic behavior in SDS-PAGE is shown in Hunfeld et al. (Ann. Hematol. 1997, 74: A87, 113; Ann. Hematol. 1998, 76: A101, P294 and Etscheid et al., Ann. Hematol. 1999, 78. : Recalls proteases isolated from PPSB concentrates not defined in detail by A42). In that case, the preparation was achieved mainly using an aprotinin matrix. As a result of the deamidation of certain peptide substrates, the activity has been described as a thrombin-like activity. Hunfeld et al. Found no effect on global blood clotting parameters such as prothrombin time, quick or platelet aggregation.

  The N-terminal sequencing of the protease described by Hunfeld et al. Is consistent with that of the protein whose cDNA was reported by Choi-Miura et al. (J. Biochem. 119: 1157-1165, 1996). In its primary structure, the corresponding protein shows homology with an enzyme named hepatocyte growth factor activating enzyme (HGFA).

Two bands isolated from SDS-PAGE under reducing conditions were subjected to N-terminal sequencing and the following agreement was established.
Molecular weight range amino acid sequence reporter of band
10-35 kDa IYGGFKSTAGK The present invention 30 kDa IYGGFKSTAG Hunfeld et al. 17 kDa IYGGFKSTAGKH Choi-Miura et al. 40-55 kDa LLESLDP The present invention 50 kDa SLDP Hunfeld et al. 57 kDa SLLESLDPWTPD Choi-Miura et al. Is also found.
Nevertheless, it is still impossible to estimate that these proteins are identical at this time. In any case, the above-mentioned proteins studied previously have not been reported to have FVIIa or other factor activation properties (see below).

  Based on its aforementioned properties, the novel protease can be used diagnostically and therapeutically.

1. Test system using novel proteases Novel proteases can be used diagnostically in test reagents. That is, the presence of Factor VII can be qualitatively and quantitatively determined in the blood coagulation test by adding a novel protease.

Conversely, this test system developed to measure FVII activation can also be used for protease detection and quantification. For this purpose, a protease-containing solution is mixed with an FVII-containing solution and both FVIIa produced after an appropriate incubation time are quantified. This can for example be carried out using Staclot ^(R) FVIIa-rTF test (Stago / Boehringer Mannheim). With the preferred procedure, this test is not limited to the concentration of FVII supplied. When knowing the amount of protease in the total protein ratio type, the ratio is:
-During pure protease preparation, by the Kieldal method or other protein assays well known to those skilled in the art, or-for example specific antibody-based antigen tests and suitable immunochemical assays such as ELISA Can then be used to determine the specific activity of the protease preparation in a corresponding manner.

  Surprisingly, with further elucidation of the properties of proteases, new properties have been found that allow additional measurements. In relation to incubation of the protease with blood clotting factors VIII / VIIIa and V / Va and subsequent quantification, it is clear that the blood clotting factor is inactivated in a manner independent of the protease concentration and the length of incubation. It was made.

Another part of the subject of the present invention is therefore a novel test system that qualitatively and quantitatively detects proteases that activate blood coagulation factor VII, in which the protease is blood coagulation factor VIII It can be quantified by its activity to inactivate / VIIIa or V / Va factor. This test system involves incubating a protease-containing solution with Factor VIII / VIIIa or V / Va and determining the amount of residual Factor VIII / VIIIa or the amount of residual Factor V / Va by conventional activity tests. It is based on an operation that measures and then determines the amount of protease by comparison with a standard curve. In carrying out this test, inhibiting the incubation of protease activity for a predetermined period of time by the addition of a limited amount of aprotinin has the advantage that these concentrations do not affect subsequent measurements of the test system. Thereafter, the residual activity of the blood clotting factor is measured by a test method well known to those skilled in the art. For this purpose, a so-called Coamatic ^(R) Factor VIII test (Chromogenix AB) is used which mainly contains Factors IX and X and quantifies the amount of FXa obtained by conversion of a chromogenic substrate in the presence of a thrombin inhibitor. In particular, the value of the test system has been proven in terms of availability (see description in [0007]). On the other hand, this amount is proportional to the amount of FVII or FVIIa. Then, if the remaining FVIII activity is determined, the concentration of protease present can be reduced.

  The degradation of FVIII / FVIIIa or FV / FVa by proteolytic action can be clearly demonstrated by SDS-PAGE. Depending on the time of incubation of the protease with eg FVIII concentrate, the bands typical for FVIII disappear, while other new bands are generated or the intensity of weak bands increases. Thus, protease activity can also be correlated by quantifying the attenuation or enhancement of the band so that it can be measured quantitatively, for example using a protease preparation. The intensity of the band in the SDS-PAGE electropherogram or the change in intensity of the band by other electrophoretic methods can be quantified using, for example, a scanner and an appropriate program well known to those skilled in the art. In addition, the antibody against the blood coagulation factor can be used for Western blotting and can be employed for evaluation in the above-described method. Particularly suitable are antibodies that specifically detect attenuated bands, or in particular generated bands. In this context, these antibodies can also be used for the establishment of other immunochemical tests, for example ELISA.

  The proteolytic inactivation described in the case of FVIII / FVIIIa is also observed when proteases are incubated with factor V / Va that exhibits some structural homology with FVIII. Degradation can be monitored in a suitable activity test system and SDS-PAGE / Western blotting.

  Despite the inactivation of FV and FVIII, the addition of proteases to blood, platelet-enriched plasma or plasma now shortens the blood clotting time, i.e. the outstanding procore in various so-called "comprehensive blood clotting tests" It was found to exhibit gurant activity. These test systems are understood to be, for example, non-activated partial thromboplastin time (NAPTT), prothrombin time (PT) and recalcification time. For example, in so-called blood coagulometers, the shortening of these times, as measured by a thrombus elasticity recorder or otherwise in a chromogenic test, correlates with the concentration of the blood coagulation promoting substance, so the concentration of that substance in the sample is Inversely, using a calibration curve of blood clotting time can be inferred. The concentration of “FVII activator” can also be determined using a selected global blood coagulation test.

  “FVII activators” likewise provide effective activators of single chain urokinase (scuPA, single chain urokinase plasminogen activator) and single chain tPA (sctPA, single chain tissue plasminogen activator) It has also been surprisingly found that it can act as a plasminogen activator activator (PAA). The activity of activated PA can be measured, for example, using chromogenic substances. That is, this property can therefore be used for the detection and quantification of “FVII activators”. Activation of the plasminogen activator can also be determined in the coupling reaction by the formation of plasmin itself in the presence of plasminogen or by lysis of fibrin clots caused by plasmin.

  Therefore, in summary, it can be said that the protease can be both detected and quantified by incubating it with a solution containing FVIII or FVIIIa and then the residual amount of FVIII / FVIIIa by a suitable activity test. In the same way, FV or FVa can be incubated with the protease and the residual amount of FV / FVa can then be quantified. The unknown protease concentration can be determined quantitatively by comparing the increase in the amount of protease included in the test with a standard curve. Various comprehensive blood clotting tests are equally suitable for quantification, and the protease concentration is read from the calibration curve based on the shortened clotting time. The PAA activity of the protease can also be used for quantitative purposes.

The characteristics of these tests are particularly good when high concentrations of calcium with suitable FV and FVIII inactivation and PAA activity are present, preferably> 0.001 mM, particularly preferably> 0.005 mM in the form of CaCl _2. To be demonstrated. As noted above, inactivation of Fv / FVIII is not promoted by heparin, as opposed to direct chromogenic assays where both heparin and heparin-like substances and calcium also increase protease activity, or Only non-significant promotions are observed. In contrast, PAA activity is stimulated in the presence of any substance, ie calcium and / or heparin or heparin-like substance.

Protease-inducing reactions can be carried out in the presence of an inhibitor, particularly heparin or heparin-like substance (preferably in the presence of heparin) antithrombin III, C1-esterase inhibitor, α ₂ -antiplasmin, inter α-trypsin inhibitor or known low molecular weight can be very efficiently attenuated or prevented by incubating the guanidino caproic acid -p- ethoxycarbonyl phenyl ester, available as protease inhibitors e.g. trade name FOY ^(R). These substances can therefore be used to stop the reaction, for example in order to accurately determine the incubation time or to further increase the specificity of the test. If the free calcium ions in the mixture are reduced, for example with a chelating agent, they can also be used for this purpose.

2. Stabilized preparation of factor V and factor VIII From the above observations on the proteolytic activity of novel proteases against blood clotting factors V and VIII, the challenge of inhibiting or attenuating protease activity is the loss of production and This occurs to avoid the formation of fragments, presumably fragments of interfering proteins. This is all the more important since FV and FVIII are usually prepared from cold precipitates obtained from plasma in the presence of calcium ions, the latter being required to maintain protein conformation.

  Another part of the subject matter of the present invention is therefore an FV free of factor V and factor VIII fragments formed by proteolysis as a result of the fact that the protease that activates blood coagulation factor VII is inhibited. This is a stabilized preparation of FVIII. Further detailed investigations have shown that inactivation of factor V and factor VIII by the proteases occurs efficiently, particularly in the presence of calcium ions at a concentration of 0.5 mM or higher. Adjusting the concentration of calcium ions in factor V or factor VIII preparations to less than 1.0 mM, preferably less than 0.5 mM, to inhibit factor activating proteases, can reduce factor V and factor VIII preparations. It can be stabilized efficiently. Although the protease inactivation of factor V and factor VIII is significantly reduced at these concentrations, the amount of calcium ions is still sufficient to stabilize the conformation of the Fv and FVIII molecules. The amount of calcium ions described above should not be excessive not only in the final product, but also in the cryoprecipitate itself and in subsequent purification steps.

  Due to the above-mentioned affinity of proteases or proenzymes for heparin and heparin-like substances, protease / proenzymes can be removed from FVIII or FV containing solutions by incubating with immobilized heparin or other immune or affinity adsorbents. Polyclonal or monoclonal antibodies useful for the preparation of immunosorbents, each antibody fragment, can be readily obtained by techniques known in the art using the whole or part of a protease or proenzyme as an antigen.

However, in order to prevent FV or FVIII proteolysis, a natural or synthetic protease inhibitor can be used as appropriate in addition to the decrease in the amount of calcium ions. Proteins such as aprotinin, α ₂ -antiplasmin, C 1 -esterase inhibitor or intertrypsin inhibitor can also be used as inhibitors. Low molecular weight substances known to those skilled in the art as synthetic serine protease inhibitors can also be used in this context. Inhibitors such as antithrombin III whose inhibitory intensity is increased by heparin or heparinoids can be added as well. That is, it has been surprisingly found that heparin itself can increase the amidolytic activity of proteases against small chromogens, but it does not support inactivation of FV / FVIII.

3. Drugs composed of novel proteases Novel proteases and / or proenzymes can be used therapeutically.
They can be used alone or in combination with substances that increase the activity of proteases, such as heparin or heparin related substances, such as heparin sulfate, and / or calcium ions. It is also possible to add factor VII to this drug as well. The use of such agents utilizing their FVIII alternative pathway activity (FEIBA) is tolerated, for example, due to the presence of antibodies, for example against FVIII and / or FIX and / or FXI and / or contact phase proteins such as FXII. It can be applied when there is a presence of other types of defects. In this connection, FVII can be activated by action on either in vitro, plasma, concentrated fractions or on purified FVII. The novel blood coagulants can also be used ex vivo to prevent systemic bleeding or to stop bleeding.

  On the other hand, the observed inhibition of novel proteases by aprotinin or the above-mentioned inhibitors can be used for the development of drugs consisting of protease inhibitors with reduced ability to clot blood. In addition, the novel proteases can also be used to identify physiological and non-physiological factors such as synthetic peptides that impair blood clotting by their protease inhibitory action. Particularly efficiently transformed chromogenic substrates such as the peptide sequence of S2288 (see above for details) can be used as the structural basis for this. If these preparations do not contain proteolytic activity, the addition of appropriate inhibitors to the clot preparation may also be necessary during their preparation.

  As the properties of proteases have been further elucidated, properties have been discovered that open up the potential for additional uses as so-called “Factor VII activators” proteases. Incubating a single-stranded plasminogen activator, such as pro-urokinase (single-stranded urokinase, sucPA, single-stranded urokinase plasminogen activator) or sctPA (single-stranded tissue plasminogen activator), "factor VII activator "Leads to the activation of these plasminogen activators (PA). In this connection, proteolysis of single-stranded PA has limitations and results in the formation of double-stranded proteases, which are particularly suitable for plasminogen activation. The plasmin produced is a fibrinolytic effector, a physiological system involved in thrombus dissolution. PAs such as prourokinase or tPA are endogenous proteins that are released as needed and activated by plasmin or kallikrein (scuPA) as is known. The mechanism by which scuPA is activated in the healthy state has not yet been fully elucidated.

  Plasminogen activators can be used therapeutically as isolated or recombinantly prepared proteins in pharmaceutical formulations associated with thrombotic diseases or complications such as femoral vein thrombosis, myocardial infarction or stroke.

  Due to the nature of the “factor VII activator” discovered this time, the latter can be used for in vivo or ex vivo activation of plasminogen activators such as urokinase or sctPA. This activity can also be applied to the use of this protease for the prevention or treatment of thromboembolic diseases as well, particularly in combination with single- or double-stranded plasminogen activators or anticoagulants. This possibility of use is consistent with the fact that proteases can also act in a procoagulant manner. The question of which of the two responses takes precedence is probably solved by the availability of physiological substances. According to current knowledge, Factor VII is moderately activated in plasma and is constantly maintained at a concentration so that sudden vascular injury can be readily addressed. On the other hand, tissue plasminogen activator as well as urokinase plasminogen activator are present only in nanogram quantities in 1 ml of plasma. The concentration is increased only by fibrin deposition or thrombus, which is increased by secretion or synthesis of plasminogen activator, which is then activated locally, especially when they bind to the thrombus, Activates plasminogen and exerts its thrombolytic action. When single-stranded PAs are present, their activation exceeds that of FVII, particularly in a locally limited manner, thereby allowing adjustment to the physiological state. Thus, this protease may also be applied to substitution by proteases and / or proenzymes in cases of congenital or acquired deficiency conditions by regulating hemostasis.

  Accordingly, another part of the subject matter of the present invention is a pharmaceutical formulation consisting of an amount of blood coagulation factor VII activating protease and / or its proenzyme form, sufficient to dissolve fibrin-containing thrombi. The formulation may further contain a single chain plasminogen activator (PA) and / or an anticoagulant. If a proenzyme is present, it is also advantageous to use a suitable activator in or with the pharmaceutical formulation described above.

  The plasminogen activator potentiating action of “FVII activator” was found to be promoted in particular by calcium and / or heparin and heparin-like substances such as dextran sulfate. Pharmaceutical preparations containing substances can be used advantageously to dissolve fibrin-containing thrombi according to the invention. In this context, the protease / proenzyme, either alone or in combination with single-stranded or double-stranded plasminogen activator, optionally exerts a specific affinity for the protease, thereby reducing the plasma half-life. It can be used as an extended carrier material or as a mediator to the surface, along with a substance that increases its activity.

  A pharmaceutical preparation comprising a blood coagulation factor VII-activating protease can be used for the treatment of diseases caused by fibrin-containing thrombi due to its specific fibrinolytic action. The fibrinolytic process is also involved in the wound healing process. In this connection, the protease and / or proenzyme can be administered intravenously or locally, subcutaneously, intradermally or intramuscularly or in the case of injury or wound, or bound to a suitable carrier matrix. . Any protease / proenzyme isolated from body fluids such as blood or plasma and protease / proenzyme prepared recombinantly or by gene transfer can be used in this context. Proteases / proenzymes are also suitable as components of so-called fibrin adhesives, which must not contain substances that inhibit protease / proenzymes such as aprotinin. In this case, the coagulation shortening action of protease is used.

  The protease / proenzyme can be used for congenital or acquired hemostatic defects, (generalized) bleeding, and various thrombus-related complications. When used in the treatment of bleeding, it is advantageous to add protease / proenzyme together with FVIII, if desired, and other clotting factors.

4). Method of sterilizing FVII-activated protease Since it is a protein isolated from human plasma, the novel protease and / or its proenzyme can only be used as a pharmaceutical preparation if it has been previously subjected to a treatment that inactivates the virus can do. The sterilization process is recognized as the most important process, especially for virus inactivation. However, the protein to be treated is required to have a stability suitable for overheating at about 60 ° C. for up to 10 hours. The optimal stabilizer for each protein must be determined separately and their concentrations optimized.
In the case of the novel protease and / or its proenzyme, the conditions for stabilizing the protein in the solution without sterilization have already been described above. In this case, a slightly acidic pH has proven particularly advantageous. However, when sterilized under these conditions, new proteases and / or their proenzymes typically lose more than 50% of their original activity.

Sterilization of a pharmaceutical preparation comprising a novel protease and / or its proenzyme requires preparation a) pH range 3.5-8.0, preferably pH range 4.0-6.8;
b) in the presence of added one or more amino acids of 0.01 mol / L or more, preferably 0.05 mol / L or more; and / or c) one added sugar or a combination of different sugars, In the presence of a concentration of 0.05 g / ml or more, preferably 0.2 g / ml or more; and / or d) a substance capable of forming a complex with the added calcium ion, such as citric acid, oxalic acid, ethylenediaminetetraacetic acid When prepared in the presence of more than a species, optimal stabilization results are guaranteed.

Additives such as albumin, Haemaccel ^(R), heparin and heparinoids, glycerol, can be used as a mixture of glycol and polyethylene glycol separately or together. After sterilization is complete, sugars, amino acids and other additives added as stabilizers can be reduced or completely removed from the preparation using methods well known to those skilled in the art. The results of the sterilization process are shown in Examples 12 and 13.

Example 1
The activation of FVII by the prepared protease was demonstrated using Staclot ^(R) FVIIa-rTF test system (Stago / Boehringer Mannheim). This detection system is based on a specific property of (recombinant) soluble tissue factor (rTF) that allows the use of preformed activated FVII (FVIIa) only to inhibit the extrinsic coagulation pathway. This makes it possible to accurately determine the actual content of FVIIa, unlike when using complete tissue factor.

  Activation experiments were performed using isolated FVII (Enzyme Research Labs). This FVII itself contains traces of FVIIa because it was isolated from human plasma. The concentration was adjusted to FVII 0.05 IU / ml by dilution with buffer. FVII was incubated with the test substance at room temperature for 10 minutes and then tested for true FVIIa content. FVIIa content was quantified using a standard curve constructed in parallel.

In preliminary experiments not described herein, aprotinin completely inhibited the activity of the prepared protease at the concentrations used, but had no direct effect on FVIIa and was significant for the FVIIa-rTF test system. It was confirmed that there was no effect.
The results listed below relate in each case to triplicate measurements. That is, the following experiment was set up as follows.

1. FVII:
Non-activated FVII was used as a control assay. This already contains traces of FVIIa (see above) in the order of FVIIa 10 mIU / ml.

2. FVII + aprotinin:
In this assay, FVII was incubated in the presence of aprotinin and the FVIIa-rTF assay was used to demonstrate that FVIIa itself is not inhibited and that the test is not affected by the aprotinin used. This was confirmed (in comparison with assay 1).

3. Protease + FVII (incubation) followed by the addition of aprotinin:
Result: FVIIa 18mIU / ml
In this case, the protease was given time to activate FVIIa. Aprotinin was simply added to plate the protease and was performed after 10 minutes incubation. The obtained FVIIa was quantified by FVIIa-rTF assay. That is, when the basal value of FVIIa (assay 1) was subtracted, FVIIa 8 mIU / ml was formed by the activity of the protease under the selected conditions.

4). Addition of protease + aprotinin and then FVII:
Result: FVIIa 11mIU / ml
In this assay, the protease was inhibited with aprotinin prior to contact with FVII. Subsequent incubation with FVII and subsequent quantification of FVIIa did not result in a significant increase in FVIIa content (range of variation in the assay, ie, 11 and 10 mIU / ml in assay 1 are not considered significant).

5). Protease:
Result: FVIIa 0mIU / ml
This assay demonstrated that at the selected concentration, the protease itself has no effect on the FVIIa-rTF test system.
In summary: From the above-the protease described activates FVII;
-Activation of FVII by proteases occurs "directly", ie independent of the presence of rTF;
-Activation of FVII is inhibited by aprotinin; aprotinin itself has no significant effect on the test system;
It is concluded that

Example 2
This example illustrates how FVII is activated in a reaction independent of the concentration of protease and the time that the protease was incubated with FVII. Test systems and reagents were selected to correspond to those described in Example 1. In the first series of experiments, the first introduced FVII was preincubated (5 minutes at room temperature) with various dilutions (1: 5, 1:10 and 1:20) of protease-containing solutions, followed by aprotinin (protease Inhibit) followed by its FVIIa content in the FVIIa-rTF assay. Again, a parallel assay was performed in which the protease was inhibited by aprotinin before contacting with FVII.

これから、ＦVIIはプロテアーゼにより、プロテアーゼの濃度に依存する様式で活性化されると結論される。 The result is given as an activation factor. That is, it corresponds to x times the value measured in the above control assay.
Assay: Protease + FVII, Incubation, + Aprotinin Control: Protease + Aprotinin, Incubation + FVII

From this it is concluded that FVII is activated by proteases in a manner that depends on the concentration of the protease.

これから、ＦVIIは、プロテアーゼによって時間依存性の様式で活性化されると結論される。 It was also demonstrated that FVII is activated by proteases in a manner that is independent of the length of incubation when the co-reactant concentration is kept constant. When an equal volume of a solution containing FVII 0.2 IU / ml and 1: 10-diluted protease solution was incubated together, aprotinin was added after the corresponding incubation period (to stop activation) and Of FVIIa was obtained.

From this it is concluded that FVII is activated by proteases in a time-dependent manner.

この実施例で用いた条件下には、プロテアーゼ活性の著しい上昇をカルシウムイオンおよび／またはヘパリンの存在下に観察することができる。 Example 3
Using this example, we will demonstrate that FVII protease activation is increased by the presence of calcium ions and heparin.
25 μl protease containing solution, 50 μl -buffer (control)
-15 mM CaCl ₂
-Heparin 50 USP units / ml
-Pathromtin (lipid mixture, aliquot dissolved according to manufacturer's instructions)
For 5 minutes at room temperature, then treated with 150 μl of tris / NaCl buffer (pH 8.2) and 25 μl of chromogenic substrate S2288 (3 mM), and the change in absorbance over time at 405 nm was measured (37 ° C.). . The activation factor (x times) relative to the buffer control is shown in the table below.

Under the conditions used in this example, a significant increase in protease activity can be observed in the presence of calcium ions and / or heparin.

Example 4
In either case, a solution containing a protease 10, 1 or 0.1 [mu] g / ml, were mixed with 25 [mu] l of FVII (2IU / ml), CaCl 2 (25mM) and 25 [mu] l of Pathromtin of 25μl ^{(R) (Dade} Behring GmbH) was added. After incubation at 37 ° C. for 0, 3, 10 and 20 minutes, 400 μl of aprotinin (500 KIU / mg) was added to stop the reaction. A sample with the initial addition of aprotinin was used as a control.

Each sample was diluted in tris buffer / BSA. In each case 50 μl of this solution was mixed with 50 μl of factor reagent (mainly consisting of FIXa, FX and thrombin inhibitors, modified according to Cosmatic® FVIII test, Chromogenix AB ⁾ and incubated at 37 ° C. for 10 minutes. After adding 50 μl of substrate (eg S2765, Na-Cbo-D-Arg-Gly-Arg-pNA), 50 μl of acetic acid (50%) is added to stop the reaction after a predetermined time incubation, Subsequently, OD _{405 nm} was measured. The concentration in the sample was determined using a standard curve of FVIII.

result:
In the first assay, the time for incubating the protease with FVIII (2 IU / ml) was kept constant (10 minutes), but the protease concentration was varied (0.1, 1 and 10 μg / ml). The reaction was stopped and the residual concentration of active FVIII was measured. As the protease concentration increased, FVIII was correspondingly inactivated (FIG. 1).
The protease content of the sample can be quantified using an appropriate standard curve.
In the second assay, the protease concentration was kept constant (10 μg / ml) and the incubation time was varied with FVIII (2 IU / ml). A significant decrease in the residual concentration of active FVIII was observed with increasing incubation time (FIG. 2).

この実施例では、ＦVがプロテアーゼによって経時的に不活性化されたことが証明される。 Example 5
The effect of “FVII activator” on the activity of factor V was examined.
25 μl protease-containing solution (0-100 μg / ml) was incubated with 50 μl FV (5 IU / ml) and 25 μl CaCl ₂ (0-20 min) followed by the addition of 400 μl buffer containing 100 KIU / ml aprotinin. .
In either case, each 100 μl incubation assay was then incubated with 100 μl FV-deficient plasma for 1 minute at 37 ° C., then mixed with 200 μl Thromborel S ^(R) and blood coagulation time on a Schnitger & Gross blood coagulometer. Was measured. The residual activity of FV was determined.
result:

This example demonstrates that FV was inactivated by protease over time.

Example 6
The effect of the “FVII activator” on the blood clotting time in the so-called comprehensive test was examined using a Schnitger & Gross blood coagulometer. All the difference values listed correspond to the blood clotting time shortened by this amount.

ＦVIIアクティベーターの添加はＮＡＰＴＴの濃度依存性の短縮を生じた。 NAPTT (non-activated partial thromboplastin time)
The protease-containing solution was diluted with buffer to 100, 30, 10, and 3 μg / ml. Each of these solutions 100 [mu] l, incubated citrated plasma 100 [mu] l (standard plasma from a pool or individual donors) and 100 [mu] l Pathromtin and ^(R) 37 ° C. for 2 minutes, then a 25 mM CaCl ₂ in 100 [mu] l was added, followed by blood The clotting time was measured. The difference between these measurements and the corresponding blood clotting time obtained using a buffer instead of protease was determined.

Addition of the FVII activator resulted in a shortened concentration dependence of NAPTT.

Plasma calcium reprecipitation time The protease-containing solution was diluted with buffer to 100, 30, 10, and 3 μg / ml. 100 μl of each of these solutions was incubated with 100 μl of citrated plasma (from standard plasma pools or individual donors) for 1 minute at 37 ° C., then 100 μl of 25 mM CaCl ₂ was added and then the blood clotting time was measured. The difference between these measurements and the corresponding blood clotting time obtained using a buffer instead of protease was determined.

PT (prothrombin time)
The protease-containing solution was diluted with buffer to 100, 30, 10, and 3 μg / ml. Each of these solutions 100 [mu] l, incubated for 1 min at 37 ° C. with citrated plasma 100 [mu] l (standard human plasma pool or individual donors) and then added ^{ThromborelS (R) (Dade Behring GmbH} ) in 200 [mu] l, followed by coagulation Time was measured.
The difference between these measurements and the corresponding blood clotting time obtained using a buffer instead of protease was determined.

Example 7
The plasminogen activator activating action of “FVII activator” was examined using single-stranded urokinase (scuPA) and single-stranded tPA (sctPA).
Assay:
0.1 ml PA solution (20 μg / ml scuPA or 100 μg / ml sctPA)
+0.1 ml test buffer or
100 U / ml heparin in test buffer or
20 mM CaCl _{2 in} test buffer
+0.5 ml test buffer +0.1 ml protease / sample (increased concentration; 2-10 μg / ml scuPA or
Is 50-200μg / ml sctPA)
Incubation at 37 ° C. +0.1 ml 100 KIU / ml aprotinin in test buffer 2 minutes incubation at 37 ° C. +0.1 ml substrate S-2444 (3 mM)
As a control, aprotinin is first introduced instead of plasminogen activator (PA) prior to the first incubation, and so on throughout each case. On the other hand, PA was not added until it was added instead of aprotinin.
The measured value difference (Δ) ΔOD _{405 nm} was optically measured. The control value obtained was subtracted from the sample / protease value and in this way the PA activity produced by PAA activity was measured (mIU / min).

表は、scuＰＡが「ＦVIIアクティベーター」の濃度およびインキュベーションの長さに依存する様式で活性化される事実を例示する。同時に、ヘパリンおよびカルシウムの両者がプロテアーゼによってもたらされたＰＡの活性化に刺激作用を示した。 result:
Activation of scuPA (20 μg / ml scuPA, 2-10 μg / ml “FVII activator”)

The table illustrates the fact that scuPA is activated in a manner that depends on the concentration of "FVII activator" and the length of incubation. At the same time, both heparin and calcium showed a stimulating effect on the activation of PA caused by the protease.

表は sctＰＡもプロテアーゼの濃度およびインキュベーション時間に依存する様式で活性化されたことを証明する。ヘパリンおよびカルシウムイオンの両者が「ＦVIIアクティベーター」のＰＡの活性化能力に対して刺激作用を示した。 Activation of scuPA (100 μg / ml sctPA, 50-200 μg / ml “FVII activator”
)
The turnover rate of activated tPA only rises by a factor of 3-4 compared to tPA proenzyme (in the case of uPA it rises by a factor of 1000-1500). A high concentration of species co-reactant had to be selected.

The table demonstrates that sctPA was also activated in a manner dependent on protease concentration and incubation time. Both heparin and calcium ions showed a stimulating effect on the ability of the “FVII activator” to activate PA.

Example 8
Two FVIII-containing solutions, one essentially free of von Willebrand factor and the other containing vWF, were incubated in the presence of the above proteases and calcium. After a predetermined time, residual FVIII activity was measured by a chromogenic test and correlated with a protease-free control assay.

For this, 25 μl of a solution containing 0.1 IU / ml FVIII was treated with the same volume of protease solution (μg / ml) and the whole was mixed with 25 μl of CaCl ₂ (25 mM). After incubation for 0, 5, 10 and 20 minutes at 37 ° C., each was treated with 400 μl of a solution containing 200 KIU / ml aprotinin to stop the proteolytic activity of the protease.
Preliminary experiments have shown that this concentration of aprotinin has no significant interference with the FVIII activity test described (assay 1 + 3). In assay 2, the protease was incubated with aprotinin prior to contacting with FVIII and then operated as described above.

In either case, 50 μl of the stopped sample (or after further dilution) was then treated with a so-called factor reagent consisting mainly of FIX, FX and thrombin inhibitors and incubated at 37 ° C. for 10 minutes. Following the addition of 50 μl of chromogenic substrate cleaved by activated FX followed by incubation for 5 minutes, 50 μl of acetic acid (50%) was added to stop the reaction and then ΔOD _{405 nm} was measured. FVIII activity (mIU) was confirmed using a standard curve prepared from the FVIII concentrate and constructed using the dilution series included in the test.

ＣaＣl₂（この場合は６.２５ｍＭ）の存在下には、ＦVIIIはプロテアーゼにより、インキュベーションの長さに依存した様式で不活性化された。ｖＷＦは、プロテアーゼによる不活性化からＦVIIIを保護しなかった。ＦVIIIと接触させる前にアプロチニンでプロテアーゼを阻害すると、前者の不活性化は防止された。 FVIII activity is expressed as a percentage of the control with no protease added.
result:

In the presence of CaCl ₂ (in this case 6.25 mM), FVIII was inactivated by the protease in a manner dependent on the length of incubation. vWF did not protect FVIII from inactivation by proteases. Inhibiting the protease with aprotinin prior to contact with FVIII prevented the former inactivation.

Example 9
In this series of experiments, Example 1 / Assay 1 was performed, but in this case the calcium concentration in the mixture of protease and FVIII was varied. For this, CaCl ₂ was added from the calcium stock solution to the final concentration shown in FIG.
result:
Reducing the calcium concentration in the assay to less than 1 mM leaves approximately 50% FVIII under these conditions. With less than 0.5 mM calcium, the residual percentage is 60% or more (FIG. 1).

Example 10
The effect of the “FVII activator” on the blood clotting time in the so-called comprehensive test was examined by the thrombus elasticity recording method.
Changes in the shear elasticity or strength of the relevant clot were recorded continuously using a Hilgard TEG meter (from Hellige). The so-called r and k values, respectively, are the time from the start of the cessation of blood and the start of the blood clotting reaction, and in the case of citrated plasma, the time of recalcium precipitation to the TEG curve extends to 1 mm, The time from the end point to the curve extended to 20 mm (clot formation time).

For this, 150 μl aliquots of blood or plasma from 5 donors were in each case incubated for 2 minutes at 37 ° C. in a measuring cuvette, and then 50 μl of sample (protease) was added and mixed. 100 μl of 25 mM CaCl ₂ was added to initiate the reaction. The final concentration of “FVII activator” in the assay was 15 μg / ml. The reduction in r time was measured for an assay containing buffer instead of sample.
result:

  This example demonstrates that in almost all cases the addition of protease causes a significant reduction in blood clotting time. In the example shown here, the fibrinolytic action of “FVII activator” was hidden in the background. This is because the “normal subject” has plasma plasminogen activator concentrations in the nanogram range and has no effect on in vitro blood clotting tests.

Example 11
The FVIII alternative pathway activity of the protease was demonstrated by the following experimental assay. That is, the thrombus elasticity recording method was used as a measurement technique. The r time was evaluated (see Example 10). Whole blood samples were incubated with a monoclonal antibody (antibody against FVIII) whose inhibitory effect on FVIII activity was known to stimulate the presence of natural FVIII inhibitor. Samples were compared to whole blood sample controls (buffer instead of Mab). Protease FEIB activity was tested by adding protease (final concentration 17 μg / ml) to whole blood samples inhibited by Mab. Furthermore, protease was added to the sample, and the effect of the protease itself on r time was measured.

抗−ＦVIII ｍＡｂによって引き起こされるｒ時間の延長はこの場合もプロテアーゼの存在により正常化され、これはプロテアーゼのＦＥＩＢ活性を例示するものである。既に上に証明したように、プロテアーゼそれ自体は血液凝固時間を短縮した。 result:

The prolongation of r time caused by anti-FVIII mAb is again normalized by the presence of protease, which exemplifies the FEIB activity of the protease. As already demonstrated above, the protease itself shortened the blood clotting time.

Example 12
To a solution containing 50 μg / ml FVII activating protease, the following substances were added to the corresponding final concentrations.
25 mM sodium citrate 25 mM HEPES
100 mM Arginine 0.75 g / ml Sucrose This solution was divided into portions and in each case aliquots were adjusted to various pH values from 5.0 to 8.6 and then heated to 60 ° C. for 10 hours.
The activity of the heated protease solution was measured by a chromogenic test and the amide degradation over time of the chromogenic substrate S2288 (HD-Ile-Pro-Arg-pHA × 2HCl, Chromogenix AB, Sweden) was recorded. This activity was expressed as a percentage of the aliquot measured in parallel without heating.

この一連の実験は、特に酸性のｐＨ範囲における安定化はプロテアーゼの不活性化を著しく低下させた。ｐＨ５.５におけるわずかな「ブレークスルー」はこの範囲にプロテアーゼの等電点があるという事実によって説明できる。クエン酸ナトリウムは好ましいｐＨ範囲で起こる＞５０％の活性の喪失を防止する。 result:

This series of experiments showed that stabilization, particularly in the acidic pH range, significantly reduced protease inactivation. The slight “breakthrough” at pH 5.5 can be explained by the fact that the isoelectric point of the protease is in this range. Sodium citrate prevents> 50% loss of activity that occurs in the preferred pH range.

Example 13
The assay at pH 6.1 (Example 1) showed the best stabilization of the protease. Therefore, various activities were evaluated by testing at pH 6.0 as described in Example 1.
The following final concentrations were set, and the protease concentration was 50 μg / ml.
50 mM sodium citrate / 50 mM NaCl, pH 6.0
0.75 g / ml sucrose 100 mM glycine 100 mM arginine

スクロースおよび各場合１種のアミノ酸の添加によってプロテアーゼの著しい安定化が証明された。 result:

The addition of sucrose and in each case one amino acid demonstrated significant stabilization of the protease.

Claims (7)

a) inhibited by the presence of aprotinin,
b) the activity is increased by calcium ions and / or heparin or heparin sulfate,
c) In SDS-PAGE, subsequent staining in the non-reducing state shows one or more bands with a molecular weight in the range of 50-75 kDa, one band with a molecular weight of 40-55 kDa and a molecular weight of 10-10 in the reduced state. One or more bands in the range of 35 kDa, and d) the band obtained in the range of molecular weight 40-55 kDa in the reduced state in SDS-PAGE has the amino acid sequence: LLESLDP and the range of molecular weight 10-35 kDa The band obtained in (1) is a protease having the amino acid sequence: IYGGGFKSTAGK, and / or the band obtained in the reduced state in SDS-PAGE has a molecular weight in the range of 60-65 kDa, and the protease containing the amino acid sequence: LLESLDP and IYGGGFKSTAGK In the test system for the qualitative and quantitative detection of B enzyme, the protease activity,
a) its activity to inactivate blood clotting factor VIII / VIIIa or factor V / Va or b) its activity to reduce blood clotting time in a comprehensive blood clotting test or c) activate plasminogen activator D) A test system that measures by its activity or its activity to activate FVII. The test system according to claim 1, wherein the activity of reducing blood clotting time is:
a) Non-activated partial thromboplastin time (NAPTT) or b) Prothrombin time (PT) or c) Plasma calcium re-deposition time or d) Activated partial thromboplastin time (APTT)
Test system to measure by. The test system according to claims 1 and 2, wherein the activity to activate and / or enhance the plasminogen activator is:
Test system measuring by activation of a) single chain urokinase PA (scuPA, single chain urokinase plasminogen activator) or b) single chain tPA (sctPA, single chain tissue plasminogen activator).   The test system according to claim 1, wherein the test system contains an amount of calcium ions of 0.001 mM or more. a) inhibited by the presence of aprotinin,
b) the activity is increased by calcium ions and / or heparin or heparin sulfate,
c) In SDS-PAGE, subsequent staining in the non-reducing state shows one or more bands with a molecular weight in the range of 50-75 kDa, one band with a molecular weight of 40-55 kDa and a molecular weight of 10-10 in the reduced state. Shows one or more bands in the range of 35 kDa, and d) a band obtained in the reduced state in SDS-PAGE in the molecular weight range of 40-55 kDa has the amino acid sequence: LLESLDP and has a molecular weight range of 10-35 kDa The band obtained in (1) is a protease having the amino acid sequence: IYGGGFKSTAGK, and / or the band obtained in the reduced state in SDS-PAGE has a molecular weight in the range of 60-65 kDa, and the protease containing the amino acid sequence: LLESLDP and IYGGGFKSTAGK Assay system using a mixture of B enzyme and appropriate proenzyme activators for testing prothrombin time replacement tissue factor / thromboplastin. a) inhibited by the presence of aprotinin,
b) the activity is increased by calcium ions and / or heparin or heparin sulfate,
c) In SDS-PAGE, subsequent staining in the non-reducing state shows one or more bands with a molecular weight in the range of 50-75 kDa, one band with a molecular weight of 40-55 kDa and a molecular weight of 10-10 in the reduced state. Shows one or more bands in the range of 35 kDa, and d) a band obtained in the reduced state in SDS-PAGE in the molecular weight range of 40-55 kDa has the amino acid sequence: LLESLDP and has a molecular weight range of 10-35 kDa The band obtained in (1) is a protease having the amino acid sequence: IYGGGFKSTAGK, and / or the band obtained in the reduced state in SDS-PAGE has a molecular weight in the range of 60-65 kDa, and the protease containing the amino acid sequence: LLESLDP and IYGGGFKSTAGK B enzyme and appropriate proenzyme activators mixture, plasminogen activator function of the test as well as the assay system used in single-stranded plasminogen activator-type of quantitation of. The test system according to claim 1, wherein the activity of enhancing a plasminogen activator is
a) a test system that measures either using a chromogenic test or b) the formation of plasmin itself in the coupling reaction in the presence of plasminogen or the dissolution of fibrin clots caused by plasmin.

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